Caveolae are a membrane specialization that was discovered nearly 40 years ago. Extensive work on transcytosis in endothelial cells has shown that in these cells they form a novel endocytic compartment called plasmalemmal vesicles that transport molecules from the blood directly to the tissue space without merging with other endocytic pathways. Several years ago, while studying the uptake of the essential vitamin 5- methyltetrahydrofolate, we discovered that folate bound to membrane receptors is sequestered by caveolae and that this is an essential step in the efficient delivery of the vitamin to the cytoplasm. We called this process potocytosis to denote the fact that it was an endocytic process specialized for the internalization of small molecules and ions. New evidence is now emerging that caveolae are also involved in cell signaling. Most likely many of the signaling activities that originate from this organelle depend on the special environment created inside the plasmalemmal vesicle each time it forms during potocytosis or transcytosis. This grant proposal outlines research that is designed to directly test this hypothesis. There are three specific aims. First, the molecular composition of purified caveolae will be studied in detail with the goal of identifying all of the lipid and protein molecules that make up the structural and regulator components of the organelle. Proteins will be isolated from pure caveolae and sequenced. Lipids will be identified using standard biochemical methods. This knowledge will give clues about caveolae function. Second, we will study the dynamics of caveolae in live cells. We will look for inhibitors of caveolae function. The pharmacologic effects of these inhibitors will give clues about the underlying mechanisms of caveolae internalization. This will be followed by attempts to identify hormones that regulate the internalization cycle. In this section, experiments are also proposed to determine the function of caveolin, the only known molecule associated with the striated coat that decorates the inside surface of caveolar membranes. The final aim is to identify one or more signaling cascades that originate in caveolae. We have designed a series of in vivo and in vitro experiments to determine how the molecular composition of the organelle changes in response to both hormonal and mechanical stimuli. The hope is that we can identify one or more input signals that lead to the production or release of an output message from caveolae. Knowledge about the role that caveolae play in integrating diverse signals that impinge on the cell has important implications for understanding disease such as cancer and atherosclerosis as well as aspects of cellular senescence involved in the aging process. Furthermore, caveolae may turn out to be an important way to deliver therapeutic drugs to cells because of the special way that they transfer substances to the cytoplasm without involving the lysosome.